Modified plants

ABSTRACT

A method for controlling endosperm size and development in plants. The method employs nucleic acid constructs encoding proteins involved in genomic imprinting, in the production of transgenic plants. The nucleic acid constructs can be used in the production of transgenic plants to affect interspecific hybridisation.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNo. PCT/GB00/02953, internationally filed Jul. 31, 2000, which waspublished in English, and claims priority to Great Britain ApplicationNo. 9918061.4, filed Jul. 30, 1999, the disclosures of both of which areincorporated in their entirety by reference hereto.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to methods for controllingendosperm size and development, and seed viability in plants. Theinvention also relates to nucleic acid constructs for use in suchmethods, as well as modified plants per se.

[0004] 2. Related Art

[0005] Yield in crop plants where seed is the harvested product isusually defined as weight of seed harvested per unit area (Duvick,1992). Consequently, individual seed weight is regarded as a majordeterminant of yield. Most monocotyledonous plants e.g. maize, wheat,(see Esau, 1965) produce albuminous seeds—that is, at maturity theycontain a small embryo and a relatively massive endosperm. Consequently,in monocotyledonous plants, the endosperm represents a significantcomponent of seed yield. Endosperms accumulate and store diversesubstances, including starch, proteins, oils and fats.

[0006] Therefore, in monocotyledons increasing the size of the endospermor its ability to accumulate storage products is likely to increaseindividual seed weight and perhaps total yield.

[0007] Endosperms are utilised commercially in diverse ways, eitherindirectly as part of the whole seed or directly following theirpurification, or the purification of certain of their constituents.Hence endosperms may represent either a proportion or the entirecommercial value of a crop. Examples of indirect usage include foddermaize and whole wheat flour. An example of direct usage of the completeendosperm is in the production of white flour for bread-making. Finally,maize oil represents an example of the utilisation of a constituent ofthe endosperm, but there are many others.

[0008] In contrast to monocotyledons, most dicotyledonous plants, e.g.oil seed rape, soybean, peanut, Phaseolus vulgaris (e.g. kidney beans,white beans, black beans), Vicia faba (broad bean), Pisum sativum (greenpea), Cicer aeietinum (chick pea), and Lens culinaris (lentil) produceexalbuminous seeds—that is, mature seeds lack an endosperm. In suchseeds the embryo is large and generally fills most of the volume of theseed, and accounts for almost the entire weight of the seed. Inexalbuminous seeds the endosperm is ephemeral in nature and reachesmaturity when the embryo is small and highly immature (usuallyheart/torpedo stage). Commonly embryo development depends on thepresence of the endosperm, which is generally accepted to act as asource of nutrition for the embryo.

[0009] Scott et al (1998) showed that the size of the endosperm in termsof the number of endosperm cells at maturity in the dicotyledonous plantArabidopsis thaliana, a close relative of oil seed rape (Brassicanapus), is positively correlated with the weight of the mature seed.Plants that developed seeds with 80% smaller endosperms (average=80nuclei) compared to controls (mean of 2x-2x (diploid plant crosses) and4x-4x (tetraploid plant crosses)=400 nuclei) produced seeds that were46% smaller (in weight terms=14 μg) than the controls (mean of 2x-2x and4x-4x=30 μg). In contrast, plants that developed seeds with 160% biggerendosperms (average=640 nuclei) compared to controls (mean of 2x-2x and4x-4x=400 nuclei) produced seeds that were 180% larger (in weightterms=54μg) than the controls (mean of 2x-2x and 4x-4x=30 μg).Arabidopsis seed in common with most other dicotyledonous seed iscomposed almost entirely of embryo. Hence the change in seed weight isalmost completely due to a change in embryo weight.

[0010] Consequently, modifying endosperm size, in terms of the number ofcells at maturity, has a dramatic impact on seed weight in seeds that donot contain endosperm at maturity. Without being bound by the following,one reasonable hypothesis is that a larger endosperm accumulates agreater quantity of reserves from the seed parent, perhaps by acting asa stronger “sink”. These reserves then provide more resources forutilisation by the growing embryo, resulting in a larger seed.Alternative mechanisms might operate, however.

[0011] The seeds of dicotyledons, like those of monocotyledons areutilised in diverse ways. For example, pulses such as soybean, peanut,Phaseolus vulgaris (e.g. kidney beans, white beans, black beans), Viciafaba (broad bean), Pisum sativum (green pea), Cicer aeietinum (chickpea), Lens culinans (lentil) are important world crops that are useddirectly for animal and human consumption. Seeds of oil rape, sunflowerand linseed are processed to produce oils.

[0012] Clearly, despite the differences in the structure of monocot anddicot seeds, particularly with respect to the presence or absence ofendosperm in mature seeds, the size of the endosperm is an importantfactor in determining individual seed weight, and therefore potentiallytotal crop yield in plants where seed is the economic harvest. Indeed,Hannah and Greene (1998) showed that maize seed weight is dependent onthe amount of endosperm ADP-glucose pyrophosphorylase, the enzymeresponsible for supplying substrate for starch synthesis.

[0013] However, there is some evidence that an increase in seed weightis associated with a reduction in seed number in many breedingpopulations. Consequently, increasing individual seed size may notresult in an increase in total yield. While maize breeding programmeshave been successful and genetic improvement has played a significantrole in increased maize yields, the genetic component to yield has ledto only a doubling of this parameter since the 1930s (Duvick, 1992). Theincrease in maize yields is currently less than 1% per year.

[0014] The genetic basis for the resistance to increased seed weightencountered in conventional breeding programmes is not understood.However, Giroux et al (1996) showed that a single gene mutation in theendosperm specific gene shrunken2 of maize resulted in a seed weightincrease of 11-18% without a reduction in seed number. This suggeststhat yield improvements are possible in a plant with a long history ofintensive and successful breeding efforts, and may therefore begenerally achievable. Similarly, Roekel et al. (1998) showed thatintroduction of the tzs gene into Brassica napus results in asignificant increase in seed yield accounted for by increased seednumber per silique and increased seed weight.

[0015] There is also evidence that seed size (weight) is positivelycorrelated with a number of components of “seed quality” such as percentgermination (Schaal, 1980: Alexander and Wulff, 1985; Guberac et al,1998); time to emergence (Winn, 1985; Wulff, 1986); durability (survivalunder adverse growing conditions) (Krannitz et al, 1991; Manga andYadav, 1995); growth rate (Marshall, 1986) and yield (Guberac et al,1998). Seed quality is an important factor in the cost of production ofcommercial seed lots since these must be tested before sale.Consequently, increasing total seed weight, even without increases intotal seed yield may have economic benefits through improvements in seedquality.

[0016] We have recently demonstrated (Scott et al., 1998) thathybridising Arabidopsis plants of different ploidies has reproducibleand dramatic effects on the weight of progeny seed. For example, aninterploidy cross between a diploid (2x) seed parent and a tetraploid(4x) pollen parent (2x-4x) results in seed which is 240% larger than2x-2x seed. Conversely, 4x-2x crosses result in a reduced seed size (60%of 2x-2x). Analysis of endosperm development in these F1 seed reveals aclear correlation between final seed size and the size of the endosperm.In common with most dicots, endosperm is not present in the matureArabidopsis seed but is required to nourish the developing embryo.Therefore, increased endosperm size translates into increased seed sizeby increasing embryo size, presumably by accumulating and then supplyingincreased nutrition, or by some other less direct means enabling theembryo to accumulate more resources from the mother.

[0017] In wild type 2x-2x crosses the endosperm is triploid and isformed by the fertilisation of a pair of fused haploid polar nuclei ofmaternal origin with a haploid sperm of paternal origin. Consequently,there is a 2:1 ratio of maternal to paternal genomes in the normalendosperms. An excess of paternal genomes in the endosperm, e.g. as aresult of a 2x-4x cross, causes increased endosperm proliferation(hyperplasia). An excess of maternal genomes in the endosperm (4x-2xcrosses) has the opposite effect: decreased endosperm proliferation(hypoplasia).

[0018] Scott et al (1998) explain these observations in terms of thegenomic imprinting (inactivation) of genes that contribute to endospermvigour, either positively or negatively. Accordingly, paternal gameteshave an overall positive effect on endosperm growth because genes thatinhibit endosperm growth or functionality are imprinted, whilst genesthat have a positive effect escape imprinting and are active in theendosperm. Adding more paternal genomes into the endosperm via atetraploid pollen parent therefore increases the number of stimulatorygenes resulting in a larger endosperm. Maternal genomes have theopposite effect. Importantly, imprinting effects have been recorded in awide range of plant species including maize and brassicas. In mammals, anumber of genes that influence foetal growth (typically expressed in theplacenta) also exhibit uniparental expression due to imprinting duringgametogenesis. Extra doses of these genes also have dramatic effects onembryo size.

[0019] Hybridisation is recognised as an important process for producingoffspring having a combination of desirable traits from both parents.Hybridisation may be interspecific or intraspecific. Interspecifichybridisation is used for introducing desirable traits such as diseaseresistance into crop species. However, the ability to make successfulsexual crosses is frequently restricted to closely related speciesbecause of the existence of a variety of prefertilisation andpost-fertilisation reproductive barriers (see Stoskopf, Tomes andChristie, 1993). One type of post-fertilisation barrier is associatedwith poor or disrupted endosperm development (post-fertilisationendosperm development barrier), which results in non-viable seed (seeEhlenfeldt and Ortiz, 1995). Endosperm failure in unsuccessful crossesis due to the operation of a genetically determined system known asendosperm dosage (Haig and Westoby, 1991). Endosperm dosage is a form ofgenomic imprinting. The removal of the endosperm dosage barrier tosexual interspecific hybridisation would have economic benefits, sincenon-sexual techniques for hybridisation e.g. somatic hybridisation arecostly and difficult.

[0020] The endosperm dosage system may also prevent intraspecifichybridisation where the parents are of different genomic constitutions(ploidies) (Haig and Westoby, 1991).

[0021] The occurrence of successful intra- and interspecifichybridisation can also be problematic. In particular, hybridisationbetween genetically modified crop plants and non modified cultivated orwild plants thereby creating hybrids carrying transgenes with thepotential for environmental and other damage inherent in this form of“transgene escape”, has caused alarm within both the public and theregulatory authorities.

[0022] There are various strategies that might be used to preventtransgene escape from crops into the wider environment. Critically, arange or spectrum of methods should be available to meet practicalconstraints imposed by the requirements of plant breeders and seedproducers and the life histories of specific crop species when in thehands of farmers. For example, the complete elimination of flowering isacceptable in vegetable crops and forage grasses during the ‘croppingstage’, but unless this trait is conditional in some way, the productionof seed by the seed producer, or the breeding of new varieties by theplant breeder, is rendered difficult or impossible.

[0023] In crops where the harvest is a fruit or a seed, given that mostcrop species are self-pollinating, the production of pollen, by at leastthe majority of flowers, is essential. Most of the major crops fall intothis category.

[0024] Cleistogamous plants produce flowers that develop normally butwhich fail to open. Consequently, self pollination occurs, but no pollenescapes from the flower. Whilst this the implementation of this solutionwould ‘only’ require modifications to flower design, such an approachmight be criticised on the grounds that pollen could escape from damageflowers.

[0025] The production of viable sexual hybrids occurs within species(intra-specific hybridisation) or between species (inter-specifichybridisation). However, in the case of inter-specific hybridisation, asuccessful outcome—viable hybrid seed—is usually only possible betweenclosely related species. Two main barriers prevent hybridisation betweenmore widely diverged species—inter-specific incompatibility at thestigma surface or within the style, which prevent fertilisation, andpost-fertilisation barriers which cause seed abortion, usually throughfailures in endosperm development (Brink and Cooper, 1947; Ehlenfeldtand Ortiz, 1995).

[0026] Brink and Cooper (1947) working in Lycopersicum were the first topropose that the primary reason for the failure of inter-specificcrosses was the same as for intraspecific crosses, namely failure of theendosperm itself The operation of this type of barrier has been reportedin numerous species including the Brassicas (see Haig and Westoby,1991). These authors and others (see Ehlenfeldt and Ortiz, 1995) alsoproposed that endosperm failure in inter-specific crosses is due to aneffective, rather than actual, imbalance in the normal ratio of maternalto paternal genomes in the endosperm. Different species are proposed tohave different genomic strengths. Hence a cross between plants of thesame ploidy may fail because the relative genomic strengths of theirrespective genomes result in a lethal effective genomic imbalance withinthe hybrid endosperm. Likewise a cross between two plants of differentploidies may succeed provided their relative genomic strengths result ina hybrid endosperm with a balanced genomic constitution. The setting ofgenomic strength is proposed to involve genomic imprinting, although theexact nature of the relationship is not understood.

[0027] In summary, the failure of intraspecific (interploidy) crossesand crosses between species may have a common cause—a genomic imbalancewithin the endosperm mediated by genomic imprinting. Modifying thegenomic strength of one or both of a pair of species that normallyhybridise may have application in generating a lethal relative endospermimbalance, thereby creating a post fertilisation barrier between the twospecies. The same approach may have application in providing apost-fertilisation barrier within a species, for example betweengenetically-engineered crop varieties and non-engineered varieties.Practically, for transgene containment the genomic strength of the cropcould be modified to prevent cross hybridisation with any problematicclose relatives. Such a technology would facilitate the exploitation ofgenetically modified plants, with considerable economic andenvironmental benefits.

[0028] There is currently considerable research effort to developtransgenic technologies (see Koltunow et al., 1995) to introduceapomixis into crop species. In natural apomictic plant species 2n seedis produced without fertilisation of the egg. The genetic constitutionof the embryo is therefore identical to that of the seed parent. Theeconomic benefits of introducing an apomixis system into crop speciesinclude true breeding F1 hybrids. Currently, F1 hybrid seed is producedannually by hybridising two genetically distinct parents in a labourintensive and costly process. True breeding (apomictic) F1 hybrids couldbe propagated for sale without the hybridisation step. The removal ofthis step would potentially therefore reduce production costs.

[0029] An essential aspect of apomixis is that the embryo is derivedfrom a cell with an unreduced (2n) number of chromosomes. In naturalapomicts this is achieved by modifying meiosis (meiotic reconstitution)such that 2n gametes are produced, or deriving the embryo from a somaticcell with the 2n number of chromosomes. Irrespective of the origin ofthe embryo the endosperm is invariably derived via meiosis which iseither restitutional or reductional. In the former case the two polarnuclei, which upon fertilisation produce the endosperm, are 2n and inthe later case n. Given that natural apomicts utilise endospermsgenerated in this way it is likely to be the case for geneticallyengineered apomictic crop plants.

[0030] A potential problem in the development of apomictic crop species,given this likely dependency on ‘sexual endosperms’ (formed byfertilisation), is ensuring the successful development of the endosperm,since the endosperm is required to nourish the embryo or itselfrepresents the principal economic harvest. One barrier to endospermdevelopment is the endosperm dosage system. In species with an endospermdosage system the ratio of maternal to paternal genomes in the endospermis 2:1. Deviation from this ratio results in endosperm abortion and seedlethality (Haig and Westoby, 1991). Natural apomicts have adopted anumber of strategies to ensure endosperm development. A few species(autonomous apomicts) develop a gynogenetic endosperm (maternal) in theabsence of fertilisation of the polar nuclei. The majority however,retain fertilisation of the polar nuclei and maintain a 2:1 genomicratio by modification of either male meiosis (to produce unreducedgametes) or the fertilisation process e.g. fertilisation involves only 1polar nucleus. Still other species successfully deviate from the genomic2:1 ratio.

[0031] For engineered apomixis the most attractive solution for ensuringendosperm development is the provision of autonomous endospermdevelopment. Solutions involving fertilisation of the polar nuclei arelikely to complicate the delivery of apomixis, for example bynecessitating the introduction of a mechanism to prevent fertilisationof the “egg” or the need to devise ways to produce 2n male gametes, orby some other means ensure a 2:1 genomic ratio.

[0032] One approach to developing an autonomous apomict involves theinduction and isolation of mutant genes that condition endospermdevelopment in sexual species without fertilisation. Extensive screeningefforts in Arabidopsis met with limited success having identifiedseveral mutant genes that condition only limited endosperm developmentin the absence of fertilisation (Ohad et al., 1996; Chaudhury et al.,1997; Ohad et al., 1999; Kiyosue et al., 1999; Luo et al., 1999). Onepotential explanation is that these mutations trigger endospermdevelopment but do not overcome the effects of the endosperm dosagesystem. Endosperms in the mutants would have a genetic constitution of 2maternal:0 paternal genomes, which deviates from the normal 2:1 genomicratio. Significantly, Scott et al, 1998, recently showed thatArabidopsis possesses a dosage system capable of causing seed abortionwhere the ratio of parental genomes in the endosperm deviatessignificantly from 2:1.

[0033] Autonomous apomixis would enable the crop to produce seed withoutany requirement for pollen. Hence transgene escape through pollen couldbe prevented by arranging for the crop plant to carry any form of malesterility that stops the production or release of functional pollen.

[0034] The interploidy cross effect on seed size, the post-fertilisationendosperm development barrier to interspecific hybridisation and thebarrier to autonomous endosperm development are all explicable in termsof genomic imprinting.

[0035] In mammals, a number of genes that influence foetal growth(typically expressed in the placenta) exhibit uniparental expression dueto genomic imprinting during gametogenesis. Extra doses of these genescan have dramatic effects on embryo size (Solter, 1998). Genomicimprinting also prevents the development of gynogenetic or androgenetic(two paternal genomes, no maternal genome) embryos (Solter, 1998).

[0036] In mammals, genes selected for imprinting are maintained in aninactive state by DNA methylation. The enzyme responsible is DNAmethyltransferase (MET) which is encoded by a single gene. Mice embryoscontaining an inactive DNA methyltransferase gene die at an earlydevelopmental stage and express both parental copies of genes that arenormally imprinted (i.e. uniparentally expressed) (Li et al, 1993). Thisdemonstrates the involvement of DNA methyltransferase in genomicimprinting and a requirement for imprinting in normal development.

[0037] In plants the imprinting mechanism is unknown. However, plantgenomes contain relatively large amounts of the modified nucleotide5-methylcytosine (Gruenbaum et al, 1981). Despite evidence implicatingcytosine methylation in plant epigenetic phenomena, such as cosupressionand inactivation of transposable elements (Napoli et al, 1990; Bender etal, 1995; Brutnell and Dellaporta, 1994, Martienssen et al., 1995;Matzke and Matzke, 1995) the role of cytosine methylation in plantdevelopmental processes and genomic imprinting remains unclear.

[0038] To date three different genes have been found that may beimprinted in the maize endosperm: tubulin (Lund et al 1995), a storageprotein regulator gene dzr (Chaudhuri, and Messing, 1994) and the r genetranscription factor that regulates anthocyanin biosynthesis (Kermicleand. Alleman, 1990). In each case, the maternally inherited allele isundermethylated, overexpressed or both, whereas the paternally inheritedallele is more methylated or has a reduced level of expression.

[0039] In Arabidopsis, ddm mutants (decrease in DNA methylation) havebeen isolated with reduced levels of cytosine methylation in repetitivesequences, although the mutations do not result in any detectable changein DNA methyltransferase activity (Vongs et al., 1993; Kakutani, 1995).After several generations of self pollination, ddm mutants exhibit aslight delay (1.7 days) in flowering, altered leaf shape, and anincrease in cauline leaf number (Kakutani, et al, 1995). Repeated selfpollination of ddm mutant plants does however result in the appearanceof severe developmental abnormalities (Kakutani et al, 1996).

[0040] Arabidopsis plants expressing a DNA methyltransferase 1 (Met1)antisense (Met1as) gene contain reduced levels of DNA methyltransferaseactivity and a correspondingly reduced level of general DNA methylation(Finnegan et al, 1995; Ronemus et al., 1996). In contrast to ddmmutants, Arabidopsis plants expressing a Met1 as gene develop variousdevelopmental abnormalities at high frequency and without repeatedself-fertilisation, including floral abnormalities (Finnegan et al,1996). PCT/US97/13358 also reports that Arabidopsis plants expressing aMet 1 as gene alter the rate of development of the plant. Thedevelopment of the endosperm in ddm mutants and plants expressing Met1ashas not been reported.

[0041] The present invention is based on the unexpected observation thata decrease of about 90% in the amount of methylated DNA present in aplant genome results in the production of gametes, both male and female,that behave in a manner that is consistent with the removal orattenuation of genomic imprinting. This is exemplified by the followingexperiments:

[0042] 1. Endosperm development in seeds derived from a cross between awild type 2x plant, as seed parent, and a 2x Met1as plant as pollenparent (2x-2x Met1as), resembles endosperm development in seeds derivedfrom a 4x-2x interploidy cross (FIGS. 1 and 3).—the endosperm issmall/underdeveloped. The resulting seed is smaller in weight terms thanseed from control 2x-2x crosses (Table 1). Hence the male gametes from aMet1as plant behave like a female gamete from a wild type plant. Thiscan be explained by proposing the removal or attenuation of imprintingin the male gamete.

[0043] 2. Endosperm development in seeds derived from a cross between a2x Met1as plant, as seed parent, and a wild type 2x plant as pollenparent, strongly resembles endosperm development in seeds derived from a2x-4x interploidy cross between wild type plants (FIGS. 1 and 3).—thatis, the endosperm is large/overdeveloped. The resulting seed is largerin weight terms than seed from control 2x-2x crosses (Table 1). Hencethe female gametes from a 2x Met1as plant behave as a male genome of anormally methylated diploid plant. This can be explained by proposingthe removal or attenuation of imprinting in the female gamete.

[0044] 3. Reciprocal crosses between 2x Met1as and 4x wild type plantsresult in seed abortion (FIGS. 1 and 3); consequently seeds derived fromthese crosses are shrivelled and do not germinate (Table 1). Thebehaviour of the endosperm in seed generated in these crosses depends onthe direction of the cross. Where the 4x plant is the seed parent theendosperm is extremely under-developed and contains very few endospermnuclei and a very small chalazal endosperm (FIG. 1, Table 1). Incontrast, where the 4x plant is the pollen parent the endosperm of theresulting seeds is over-developed, and contains many endosperm nucleiand a very well developed chalazal endosperm with many associatedchalazal nodules (FIGS. 1 and 3, Table 1). This outcome resembles thoseobtained in crosses between 2x and 6x wild type plants which routinelyfail to produce viable seed (FIG. 3) and display very under— (6x-2x) orover-developed (2x-6x) endosperm depending on the direction of thecross. These crosses represent examples of lethal parental genomicexcesses within the endosperm that result from the large disparitybetween the ploidy level of the respective parents. The similaritybetween the outcomes and the behaviour of the endosperm in 2x Met1as-4xand 2x-6x reciprocal crosses can be explained by proposing that male andfemale gametes derived from 2x Met1as plants behave, in part, likegametes of the opposite sex with respect to genomic imprinting. Thisagain strongly suggests that DNA hypomethylation caused by the Met1 asgene removes or strongly attenuates genomic imprinting.

[0045] 4. The behaviour of plants homozygous for the ddm mutation inreciprocal crosses with 2x and 4x wild type plants is very similar tothat of plants homozygous for the Met1as gene (see FIG. 2 and Table 1).This strongly suggests that the basis of the interploidy cross effect isassociated with general DNA hypomethylation.

[0046] Thus, in a first aspect, the present invention provides a methodfor the production of modified endosperm which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression in the male or female germ line and/or gametes ofthe resultant plant and one or more sequences whose expression ortranscription products(s) is/are capable of modulating genomicimprinting.

[0047] As will be described herein, modulation of imprinting of plantgamete DNA can be used alter endosperm development. The effects can beapplied to male or female gametes of the transformed plant. Thus, in asecond aspect, the present invention provides a method for theproduction of modified endosperm which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression within the developing gynoecium, especially thecell lineage that gives rise to the female germ line (megasporocytetissue), within the ovule of the resultant plant and one or moresequences whose expression or transcription product(s) is/are capable ofmodulating genomic imprinting.

[0048] In a third aspect, the present invention provides a method forthe production of modified endosperm which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression within the developing stamen, especially the celllineage that gives rise to the male germ line (microsporocyte tissue) ofthe resultant plant and one or more sequences whose expression ortranscription product(s) is/are capable of modulating genomicimprinting.

[0049] There are a number of proteins known or suspected to be involvedin the process of genomic imprinting. Altering the rate of expression ofthose genes in the germ line of either sex can also be used to alter thedevelopment of the endosperm in a parent-specific manner.

[0050] In the African claw toad Xenopus laevis, the product of themethyl-cytosine binding protein 2 (MeCP2) has been shown to specificallybind to methylated cytosines (Kass et al., 1997; Jones et al., 1998).This protein, of which conserved homologs in mammals also exist, forms acomplex at the C-met locus with several other proteins. Amongst theseare the transcription-repression mSin3 proteins (Nan et al., 1998;Laherty et al., 1997) and a number of histone deacetylases (HDAC). Theactivity of the latter genes is presumed to be an important step in theprocess of anchoring histones to the DNA and hence the formation ofheterochromatin and the silencing of genes (reviewed in Razin, 1998 andPazin and Kadonaga, 1997). The MeCP2protein may thus constitute thefirst step in the gene silencing process by guiding theheterochromatin-forming machinery to C-met loci. Interestingly, incontrast with this the protein has also been found to have ade-methylating function in that it removes methyl-groups from cytosineresidues (Bhattacharya et al., 1999).

[0051] If the homologs of proteins in the C-met binding complex inplants are likewise involved in uniparental gene silencing (imprinting)then inactivation of these genes in the maternal or paternal germ lineswould be predicted to mimic the uniparental inactivation of the genesresponsible for methylation. In addition, there could be a cumulativeeffect if more than one gene is inactivated. If for instanceinactivation of the MET1 gene by antisense transcription or ds-RNA inone of either germ lines is not complete, then introduction of anadditional vector causing inactivation of one of the other components ofthe imprinting machinery will enhance the effect.

[0052] In a preferred aspect, the present invention provides a methodfor the production of modified endosperm based on targeting the germline or gametes with transgenes which alter the capacity of genes toform, maintain or express imprints. This can be achieved in a number ofways. Firstly, by incorporation of one or more sequences encodingproteins associated with the application or maintenance of geneticimprints. Specifically, such sequences may encode a histone deacetylase,methyl cytosine binding protein or Sin 3 proteins, for example, m Sin 3.

[0053] Alternatively, the transgene may incorporate sequences includingthe FIE gene or the FIS gene, for example fis1, fis2 or fis3.

[0054] Imprinted genes may also contain, or be located close to, signalswithin the DNA sequence (a particular nucleotide sequence motif) thatmark them out for imprinting during gamete production. Such a motif may,in addition to expressed proteins associated with the formation and/ormaintenance of genomic imprints, be involved in the formation of an“imprinting complex”. It is contemplated that removing or inactivatingthe DNA motif, or restricting the availability of the associatedproteins, in the imprinting complex may provide a means for preventingor attenuating the application of imprints, thereby allowing theexpression of genes which may otherwise be silenced in the endosperm.

[0055] The present invention further provides methods for removing orattenuating genomic imprinting, based on targeting the germ line orgametes with transgenes which alter the methylation pattern of genes, ortheir capacity to form or maintain imprints, within the developingendosperm. Thus, in a fourth aspect, the present invention provides amethod for the production of modified endosperm, which comprises thestep of transforming a plant, or plant propagating material, with anucleic acid molecule comprising one or more regulatory sequencescapable of directing expression in the male or female germ line and/orgametes of the resultant plant, and one or more sequences whoseexpression or transcription product(s) is/are capable of altering thedegree of methylation of nucleic acid.

[0056] The restriction of imprint removal or attenuation to one or othersex of gamete is desirable for 3 reasons:

[0057] 1. To provide for removal of imprinting in a single sex of gametewithin an individual plant. This will produce the asymmetry ofimprinting that is required to mimic the interploidy cross effect in aself-fertilising plant.

[0058] 2. To prevent developmental abnormalities that are associatedwith generalised hypomethylation, such as occurs with the CaMV35S drivenMet1 antisense gene.

[0059] 3. To prevent the attenuation of the interploidy cross effect dueto the expression of the hypomethylation gene (Met1as) within theendosperm. Crosses between two 2x Met1as plants result in seed with aslightly increased number of endosperm nuclei and normal seed weight(Table 1), which is most easily explained by proposing that thecombination of hypomethylated gametes of both sexes allows normalendosperm development

[0060] The important property of the nucleic acid molecule used in thetransformation step is that DNA of cells that contribute to one sex ofgerm line is subject to alteration of the pattern of DNA methylationthrough the activity of the transgenes. The germ-line is the tissuewithin the reproductive organs that produces the gametes. In the anthers(stamen) this is the microsporogenous cell tissue and in the pistil(gynoecium) the megasporocyte tissue.

[0061] Since the timing of the application of the genomic imprints iscurrently not known the activity of the regulatory sequences, e.g.promoters (or fragments of promoters) promoters should be as broad aspossible whilst remaining consistent with the principles discussedherein.

[0062] As will be described herein, alteration of the methylation ofplant gamete DNA can be used to modify endosperm development. Thus, in afifth aspect, the present invention provides a method for the productionof modified endosperm, which comprises the step of transforming a plant,or plant propagating material, with a nucleic acid molecule comprisingone or more regulatory sequences capable of directing expression withinthe developing gynoecium, especially the cell lineage that gives rise toor comprises the female germ line (megasporocyte tissue), within theovule of the resultant plant, and one or more sequences encoding one ormore proteins which cause methylation or demethylation of nucleic acid.

[0063] In this aspect of the invention, the resultant endosperm islarger, and the seed produced is heavier. Herein, suitable promotersinclude promoter fragments from the Arabidopsis AGL5 gene. (Sessions etal., 1998), the Petunia FBP7 and FBP11 genes (Angenent et al., 1995;Colombo et al., 1995), Arabidopsis BEL1 gene (Reiser et al., 1995),Arabidopsis MEDEA (FISI) gene (Grossniklaus et al., 1998; Kiyosue etal., 1999), Arabidopsis FIS 2 (Kiyosue et al., 1999), FIE (FIS3) (Ohadet al., 1999; Kiyosue et al., 1999), orthologs/homologues of these genesfrom other species. Other promoters that drive expression that isrestricted to cells within the female reproductive organs thatcontribute to the female germ line would also be suitable. Especiallysuitable are promoters from gynoecium-specific genes that are firstexpressed during early gynoecium development, preferably before thedifferentiation of individual ovules, and which maintain theirexpression until ovule differentiation is complete (contain egg cell andbinucleate central cell).

[0064] As used herein, the term “homologues” of the genes is defined toinclude nucleic acid sequences comprising the identical sequence to thegene or a sequence which is 40% or more identical, preferably though45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% to the sequence ofthe gene at the nucleic acid residue level, using the default parametersof the GAP computer program, version 6.0 described by Deveraux et al,1984 and available from the University of Wisconsin Genetics ComputerGroup (UWGCG). The GAP program utilises the alignment method ofNeedleman and Wunsch 1970 as revised by Smith and Waterman 1981.

[0065] In a sixth aspect, the present invention provides a method forthe production of modified endosperm which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression within the developing stamen, especially the celllineage that gives rise to or comprises the male germ line(microsporocyte tissue) of the resultant plant and one or more sequencesencoding one or more proteins which cause methylation or demethylationof nucleic acid.

[0066] In this aspect of the invention, the resultant endosperm issmaller, and hence the seed is lighter. Herein, suitable promotersinclude promoter fragments derived from the Arabidopsis genes APETALA3(Jack et al., 1992; Irish and Yamamoto, 1995), the ArabidopsisPISTILATTA gene (Goto and Meyerowitz, 1994), the Arabidopsis E2 (Fosteret al., 1992), the Arabidopsis APG (Roberts et al., 1993),homologues/orthologs of these genes from other species. Other promotersthat drive expression that is restricted to cells within the malereproductive organs that contribute to the male germ line would also besuitable. Especially suitable are promoters from stamen-specific genesthat are first expressed during early stamen development, preferablybefore the differentiation of individual microsporocytes, and whichmaintain their expression until stamen differentiation is complete.

[0067] Herein, promoters that drive gene expression in cells of the germline or in cells that represent the direct progenitors of the germ linewithin either the stamen or pistil and which, when in conjunction withthe Met1as gene, produce hypomethylated gametes are referred to as ‘germline’ promoters.

[0068] Thus, as will be appreciated by the skilled person, the presentinvention allows for the modification of the endosperm such that it iseither increased or decreased in size. In addition, the development ofthe endosperm can be altered such that the modified plants can be usedin carrying out intraspecific hybridisation, erecting artificialbarriers to intra- and interspecific hybridisation to prevent “transgeneescape”, or in engineering apomixis.

[0069] In one specific embodiment, the degree of methylation isincreased. This can readily be achieved by incorporating one or moresequences encoding one or more methylating enzymes into the transgene.

[0070] Examples of suitable methylating enzymes include:

[0071] i) Methylase 1 (acc. nr. C10692);

[0072] ii) Methylase 1-like gene (acc. nr. Z97335);

[0073] iii) Methylase ² (acc. Nr AL021711); and

[0074] iv) Chromomethylase (acc. Nr. U53501); all from Arabidopsis.

[0075] In another specific embodiment, the degree of methylation isdecreased. This can be achieved in a number of ways. Firstly, byincorporation of one or more sequences encoding one or moredemethylating enzymes, such as de-methylase (=MeCP2-homologue; seebelow) (acc. nr. AL021635) into the transgene. Alternatively, thetransgene can incorporate sequences which cause down regulation ofmethylating enzymes already present in the plant. For instance, one canuse antisense sequences, e.g. the Met1as “gene”. In addition, it hasbeen found that incorporation of whole or partial copies of an alreadypresent gene can result in suppression of gene expression. Thus, thetransgene can incorporate additional copies, or partial copies, of genesencoding methylating enzymes already present in the plant. In anotheralternative, the transgene can incorporate a sequence encoding aribozyme.

[0076] With respect to the sequence, or sequences capable of alteringthe degree of methylation, sequences encoding methylating ordemethylating enzymes can be used. Examples of the latter include:

[0077] 1) Methylase 1-like gene (acc. nr. Z97335);

[0078] ii) Methylase 2 (acc. nr. AL021711);

[0079] iii) Chromomethylase (acc. nr. U53501);

[0080] iv) de-methylase (=MeCP2-homologue; see below)(acc. nr.AL021635);

[0081] In Arabidopsis, possible homologs of the following genes havebeen found:

[0082] MeCP2 (acc nr. AL021635)

[0083] HDAC1/2 (acc. nr. AF014824 & AL035538)

[0084] mSIN3 (acc. nr. AC007067_(—)5 & AC002396)

[0085] _p300: a histone acetylation-gene (acc. nr. AC002986.1 &AC002130.1)

[0086] In a seventh aspect, the present invention provides an isolatedor recombinant nucleic acid molecule, eg a DNA molecule, which comprisesone or more regulatory sequences capable of directing expression in themale or female germ line and/or gametes of a plant and one or moresequences capable of altering the degree of methylation of nucleic acid.

[0087] In a preferred embodiment of the seventh aspect, the degree ofnucleic acid methylation is decreased. An eight aspect of the presentinvention provides the use of a transgene in which the degree of nucleicacid methylation is decreased, as a post-fertilisation barrier tohybridisation, for example, interspecific or intraspecific hybridisationbetween plants.

[0088] The expression “barrier” is defined to include all forms ofreproductive barrier which are associated with poor or disruptedendosperm development. Specifically, the term barrier refers to apost-fertilisation endosperm development barrier, which results innon-viable seed.

[0089] The transgene provides a barrier to hybridisation by modifyingthe genomic strength of one or both a pair that normally hybridisethereby causing an effective genomic imbalance leading to failed ordisrupted endosperm development. The genomic strength may be modified byremoving or attenuating genomic imprinting through DNA hypomethylation.The advantage of preventing hybridisation between plants of the samespecies (interspecific hybridisation) is discussed earlier in theapplication in the context of preventing transgene escape.

[0090] In a ninth aspect, the present invention provides the use of atransgene in which the degree of nucleic acid methylation is decreased,in overcoming a post-fertilisation barrier to hybridisation. In thiscontext, the barrier to hybridisation between plants of the same species(interspecific hybridisation) arises through endosperm dosage whichleads to failed endosperm development. The removal or attenuation ofgenomic imprinting through DNA hypomethylation, may remove the endospermdosage barrier to interspecific hybridisation. The removal of theendosperm dosage barrier to several interspecific hybridisation wouldhave economic benefits as discussed previously in the application.

[0091] The nucleic acid of the seventh aspect of the invention willnormally be employed in the form of a vector and such vectors form afurther aspect of the invention.

[0092] The vector may be for example a plasmid, cosmid or phage. Vectorswill frequently include one or more selectable markers to enableselection of cells transfected or transformed and to enable theselection of cells harbouring vectors incorporating heterologous DNA.Examples of such a marker gene include antibiotic resistance(EP-A-0242246) and glucuronidase (GUS) expression (EP-A-0344029).Expression of the marker gene is preferably controlled by a secondpromoter which allows expression in cells other than the gametes, thusallowing selection of cells or tissue containing the marker at any stageof regeneration of the plant. The preferred second promoter is derivedfrom the gene which encodes the ³⁵S subunit of Cauliflower Mosiac Virus(CaMV) coat protein. However any other suitable second promoter could beused.

[0093] Cloning vectors may be introduced into E. coli or anothersuitable host which facilitate their manipulation. DNA in accordancewith the invention will be introduced into plant cells by any suitablemeans. Thus, according to yet a further aspect of the invention, thereis provided a plant cell including DNA in accordance with the invention.

[0094] DNA may be transformed into plant cells using a disarmedTi-plasmid vector and carried by agrobacterium by procedures known inthe art, for example as described in EP-A0117618 and EP-A-0270822.Alternatively the foreign DNA could be introduced directly into plantcells using a particle gun. This method may be preferred for examplewhen the recipient plant is a monocot.

[0095] A whole plant can be regenerated from a single transformed plantcell, thus in a further aspect the present invention provides transgenicplants (or parts of them such as propagating material) including DNA inaccordance with the invention. The regeneration can proceed by knownmethods. When the transformed plant flowers it can be seen to be malesterile by the inability to produce viable pollen. Where pollen isproduced it can be confirmed to be non-viable by the inability to effectseed set on a recipient plant.

[0096] The present invention also provides transgenic plants and thesexual and/or asexual progeny thereof which have been transformed with arecombinant DNA sequence according to the invention. The regeneration ofthe plant can proceed by any known convenient method from suitablepropagating material.

[0097] A further aspect of the present invention provides a method formanipulating genomic imprinting in a plant, which comprises the step oftransforming a plant, or plant propagating material, with a nucleic acidmolecule comprising one or more regulatory sequences capable ofdirecting expression in the male or female germ line and/or gametes ofthe resultant plant, and one or more sequences whose expression ortranscription product(s) is/are capable of altering the degree ofmethylation of nucleic acid.

[0098] Preferred features for each aspect of the invention are as foreach other aspect mutatis mutandis.

[0099] The present invention will now be described with reference to thefollowing examples, which should not be construed as in any way limitingthe invention. The examples are accompanied by the following FIGUREs:—

[0100]FIG. 1—Embryo and endosperm development following crosses with met1-antisense expressing plants as a parent. Confocal micrographs ofFeulgen-stained seeds 4-6 days after pollination. Column 1, embryo;column 2, chalazal endosperm; column 3, peripheral endosperm. Note apaternal excess phenotype (over developed chalazal endosperm, highlyproliferated peripheral endosperm) in crosses with a demethylated plantas the mother (row 1, 2) and a maternal excess phenotype (small orabsent chalazal endosperm and a poorly developed peripheral endosperm)in crosses with a demethylated plant as the father (row 4, 5). See textfor full details.

[0101]FIG. 2—Embryo and endosperm development following crosses with ddm1-mutant plants as a parent. Confocal micrographs of Feulgen-stainedseeds 4-6 days after pollination. Column 1, embryo; column 2, chalazalendosperm; column 3, peripheral endosperm. See text for full details.

[0102]FIG. 3—Embryo and endosperm development following interploidycrosses and balanced crosses. Confocal micrographs of Feulgen-stainedseeds 4-6 days after pollination. Column 1, embryo+peripheral endosperm;column 2, chalazal endosperm. For the 2x-4x and 2x-6x crosses (row 6, 7)the peripheral endosperm is shown as an inset. See text for fulldetails.

[0103]FIG. 4—Schematic diagram showing the method of construction ofpAGL5-bin.

[0104]FIG. 5—Schematic diagram showing the method of construction ofpAP3-bin.

[0105]FIG. 6—Schematic diagram showing the method of construction ofpAGL5-asMET1

[0106]FIG. 7—Schematic diagram showing the method of construction ofpAP3-asMET1

[0107]FIG. 8—Seed production following inter-specific crosses betweenArabidopsis thaliana and Arabidopsis lyrata. Light micrographs of seedstaken from mature seed pods. A, 4x A. thaliana×A. lyrata; note seeds areshrivelled (see Table 3 for germination data). B, 4x A.thalianaMet1a/s×A. lyrata (4x A.thaliana Met1a/s=hypomethylated tetraploid lineexpressing Met1 a/s gene); note that seeds are plump (see Table 3 forgermination data). See text for full details.

[0108]FIG. 9—Seed production following inter-specific crosses betweenArabidopsis thaliana and Cardaminopsiss arenosa. Light micrographs ofseeds taken from mature pods. A, 4x A.thaliana×C. arenosa; note seedsare plump (see Table 3 for germination data). B, 4x A.thalianaMet1a/s×C.arenosa; note seeds are shrivelled (see Table 3 forgermination data).

[0109]FIG. 10—Seeds from a fie-1/FIE×FIE/FIE cross. (A) Light micrographshowing the two classes of seeds, plump (p1) and shrivelled (sh). (Bar=5mm). (B-G) Confocal micrographs of normal (B-D) and aborting (E-G) seedsat 8 DAP, centred on micropylar (B, E), central (C, F), and chalazal (D,G) regions of the embryo sac. The endosperm in (E-G) is overgrown andhas not cellularized. Bar=50 μm.

[0110]FIG. 10—Seeds from a fie-I/FIE×FIE/FIE cross. (A) Light micrographshowing the two classes of seeds, plump (p1) and shrivelled (sh). (Bar=5micrometers). (B-G) Confoccal micrographs of normal (B-D) and aborting(E-G) seeds at 8 DAP, centred on micropylar (B, E), central (C, F), andchalazal (D,G) regions of the embryo sac. The endosperm in (E-G) isovergrown and has not cellularized. Bar=50 micrometers.

[0111]FIG. 11—Seeds from a [fie-1/FIE×FIE/FIE; METI als/METI a/s] cross.(A) Light micrograph showing the two classes of seeds. All seeds areplump, indicating that a pollen parent hypomethylated by the METI a/stransgene can rescuefie-1 mutant seeds. Bar=5 mM. (B) Identification ofthe fie-1 and FIE alleles by PCR and restriction enzyme analysis. Thewild type FIE allele produces four bands (lane 1, WT) while fie-1/FIEheterozygotes (lane 2, Het) have an extra band. All large seeds scoredhad the heterozygous pattern (lane 3) while all small seeds were wildtype (lane 4). (C-H) Confocal micrographs of seeds at 8 DAP. The seed in(C-E) has a similar phenotype to seeds from interploidy crossesgenerating maternal genomic excess, while (F-H) shows characteristics ofpaternal excess (see text, and Scott. et al., 1998). Bar=50 μm.

[0112]FIG. 12—Autonomous endosperm development in unfertilised seeds ofArabidopsis thaliana.

[0113] Confocal micrographs of fertilization-independent seeds producedby emasculated fie-1/FIE heterozygotes with normal and reducedmethylation. (AC) Seed-like structure from a plant with normalmethylation. (A) Optical section showing peripheral endosperm but nowell differentiated chalazal endosperm. Bar=50 μm. (B) Clusteredendosperm nuclei at periphery. (PE, peripheral endosperm.) Bar=50 μm.(C) Endosperm at micropylar. (MP) and chalazal (CHP) poles. (D-G)Seed-like structures from fie-1/FIE; METI a/s plants. (D, E) Type Iseed-like structures at 7 (D) and 10 (E) days after emasculation (DAE).In these the endosperm cellularizes and fills the interior of the embryosac. (F, G) Type 2 seed-like structures at 7 (F) and 10 (G) DAE. Theseproduce micropylar and chalazal in addition to peripheral endosperm.

EXAMPLE 1 The use of Gametes from Hypomethylated Plants (Met1as and ddm)Mimics the Interploidy Cross Effect (Alters Number of Endosperm NucleiFormed and Consequently the Weight of Mature Seed).

[0114] Reciprocal interploidy (different ploidy) crosses between diploid(2x), and tetraploid (4x) (Scott, et al 1998) or hexaploid (6x) (Scott,et al 1998) Arabidopsis plants result in changes to both the size of theendosperm, in terms of the number of endosperm nuclei and volume of thechalazal endosperm, and to the dry weight of mature seeds (see Table 1)and the viability of the seed (Table 1). This is the interploidy crosseffect.

[0115] Crosses Involving Met1 as Plants

[0116] Intraploidy (same ploidy) crosses between 2x Met1as plants and 2xwild type plants mimic this effect (see Table 1 and FIGS. 1 and 3). Across between a 2x Met1 as plant as seed parent and a 2x wild type plantas pollen parent produces seeds with an average of 450 endosperm nuclei(an increase of 130% over 2x met-2x met cross), a relative increase inchalazal endosperm volume of 75% compared to 2x met-2x met seed, and amature dry weight of 20 μg (an increase of 33% compared to seed from 2xmet-2x met cross) (see Table 1).

[0117] A cross between a 2x wild type plant as seed parent and a 2xMet1as plant as pollen parent produces seeds with an average of 200endosperm nuclei (a reduction of 43% over 2x met2x met cross), arelative decrease in chalazal endosperm volume of 50% compared to 2xmet2x met seed, and a mature dry weight of 10 μg (a decrease of 30% overa wild type 2x met2x met cross) (see Table 1). TABLE 1 Outcomes ofcontrol crosses and crosses involving Met1 antisense and ddm mutantplants Maximum number of Relative Relative Interploidy Viability ofperipheral volume of change to Seed cross hybrid endosperm chalazalcellularisation weight Cross phenotype¹ seed (%)² nuclei³ endosperm⁴time (days)⁵ (μg)⁶ 2x-2x NA 95-100 400 1 10 22 4x-4x NA 95-100 400 2.5 036 6x-6x NA 95-100 300 3.5 0 44 2x-4x PE 95-100 640 2 +1 54 4x-2x ME95-100  80 0.6 −1 14 2x-6x PE 0⁷ 400 6.8 absent 6 6x-2x ME 0⁷  50 0.2−1.5 4 2xmet-2xm PE 95-100 350 1 0 15 2xmet-2xm   (90)⁸ (598)⁸ (13.6)⁸2x-2xmet ME 95-100 200 0.5 −0.5 10 2xmet-2xm   (93)⁸ (227)⁸ (9.5)⁸2xmet-2x PE 95-100 450 1.75 −0.5 20 2xmet-2xm (97)⁸ (1,365)⁸ (32.5)⁸2xddm-2xddm PE 95-100 350 1.25 0 19 2x-2xddm ME 95-100 250 0.5 −0.5 122xddm-2x PE 95-100 400 2 +0.5 21 4x-2xmet ME 0⁷ 100 0.3 −1.5 3 2xmet-4xPE 0⁷ 740 4.4 >+3 15 4x-2xddm ME 0⁷ 150 0.3 −1.5 5 2xddm-4x PE 0⁷ 6803.5 >+3 5

[0118] The presence and (possible) activity of the Met1 a/s gene withinthe endosperm potentially complicates the interpretation of the dataproduced in out crosses involving homozygous Met1a/s plants. In suchcrosses the endosperm (and embryo) inherit a single copy of the Met1a/s,either from the seed or pollen parent. If the Met1as is active withinthe endosperm it may,

[0119] 1. disrupt endosperm development since Met1as plants show variousvegetative and floral abnormalities associated with the mis-expressionof certain genes that regulate development (Finnegan, 1996). However,the presence of the Met1as gene does not appear to have this effectsince the endosperms of seeds derived from self pollinated Met1 asplants appear developmentally normal except for a degree of paternalexcess (FIGS. 1).

[0120] 2. attenuate the magnitude of the interploidy cross effect, bydemethylating and thereby erasing imprints from the genome contributedby the normally methylated parent. The imprints must be maintained andpropagated in the endosperm if the interploidy cross effect is to bemimicked. The removal of imprints via the action of the Met1as genecould reactivate imprinted loci such that the endosperm genomes behaveas if derived from same ploidy parents.

[0121] To demonstrate that the interploidy cross effects described aboveare due to the effect of the Met1as gene on the imprinting of gametesrather than any effect within the endosperm we present data from crossesinvolving plants hemizygous (that is carrying a single copy) of theMet1as gene. Such plants show patterns of general DNA demethylationsimilar to homozygotes. Hence gametes derived from these plants aregenerated in a hypomethylating environment, but because the plants arehemizygous only 50% of these gametes contain the Met1as gene. Thisenables gametes to be produced in a demethylating environment which thendo not subsequently contribute a Met1as into the endosperm when used incrosses. This allows the effect of removing imprints within the gametesto be evaluated in endosperms that do not contain the Met1as gene.

[0122] The results of reciprocal crosses involving hemizygotes and 4xwild type plants are shown in Table 2. Both crosses result in a 1:1ratio of plump, viable: shrivelled, inviable seed. The shrivelled seedsare assumed to result from lethal parental excess caused by the union ofa hypomethylated gamete from the hemizygote and a 2x gamete from the 4xparent. Conversely, the plump seeds are assumed to result from normallymethylated gamete from the hemizygote and a 2x gamete from the 4xparent. Met1as plants appear therefore to produce both normallymethylated and hypomethylated gametes. The plump seeds produce plantswhich segregate 1:1 for the Met1 as gene. Presumably, the shrivelledseeds also segregate 1:1 for the Met1 as gene. This data thereforedemonstrates that the presence of the transgene in the endosperm is notresponsible for the lethality phenotype associated with 2x Met1as4xreciprocal crosses. If this were the case, seeds containing the Met1asgene would not be recovered among the plump, viable seed class.

[0123] Crosses involving ddm mutant plants

[0124] Table 1 shows that crosses between wild type diploid and wildtype tetraploid plants and plants homozygous for the ddm mutation havevery similar outcomes to crosses involving plants containing the Met1asgene. The common feature of the ddm mutation and the action of theMet1as gene is that plants containing these genes have highlyhypomethylated DNA. This shows that the interploidy cross effectproduced in crosses involving gametes derived from ddm and Met1as plantsis related to DNA hypomethylation.

[0125] The hemi zygote data (Table 2) further suggests that thephenomenon involves hypomethylation of the gametes, presumably throughthe removal of genomic imprints. TABLE 2 Outcomes of reciprocal crossesbetween Arabidopsis plants hemizygous for the Met1 as gene and wild type4x plants. Mature Seed Seed phenotypes viability Proportion Seed weight(%)¹ (%)² viable seeds (μg)⁴ Plump Shrivelled Plump Shrivelled carryingMetlas Plump Shrivelled seeds seeds seeds seeds gene (%)³ seeds seeds4x-2xmetHET 50 50 95-100 0 50 11 2 2xmetHET-4x 50 50 95-100 0 50 23 8

EXAMPLE 2 Construction of Expression Cassettes that Restrict GeneExpression to either the Gynoecium or the Stamen.

[0126] Example 1 demonstrates that uniparental demethylation can be usedto control seed size. However, the increase in seed weight in the cross2x met1a/s-2x is smaller than for the corresponding interploidy cross(2x-4x). This may be due to the reduced fitness of the 35SMet1as femalelines since demethylation is approximately constitutive. In order toreduce and eliminate this effect and to allow seed size changes to beobtained in a single plant it is necessary to restrict demethylation asmuch as possible to the germ line or gametes.

[0127] a. Designing a General Female-Germ Line Specific ExpressionVector

[0128] An expression vector based on the female-specific AGL5 promoter(Sessions et al (1998)) is constructed as described below. The nos polyAsignal sequence is excised from pCaMVNEO (Fromm et al (1986)) as aBamHI, HindIII fragment and cloned between the BamHI and HindIII sitesof pBin19 (Bevan 1994) forming pNosterm-bin. A 2.2 kb AGL5 promoter isPCRed from Arabidopsis genomic DNA using the primers AGL5F and AGL5Rwhich introduce an EcoRI and a KpnI site at the ends of the AGL5 PCRfragment. 5′ CCGAATTCTTCAAGCAAAAGAATCTTTGTGGGAG 3′ AGL5F       EcoRI5′ CGGTACCTATAAGCCCTAGCTGAAGTATAAACAC 3′ AGL5R      KpnI

[0129] The AGL5 PCR fragment is cloned as an EcoRI, KpnI fragmentbetween the EcoRI and KpnI sites of pNosterm-bin forming pAGL5-bin (FIG.4).

[0130] b. Designing a General Male-Germ Line Specific Expression Vector

[0131] An expression vector based on the male-specific AP3 promoter(Irish and Yamamoto (1995)) is constructed as described below. A 1.7 kbAP3 promoter is PCRed from Arabidopsis genomic DNA using the primersAP3F and AP3R which introduce an EcoRI and a KpnI site at the ends ofthe AP3 PCR fragment. 5′ CCGAATTCAAGCTTCTTAAGAATTATAGTAGCACTTG 3′ AP3F     EcoRI 5′ GGGTACCTTCTCTCTTTGTTTAATCTTTTTGTTGAAGAG 3′ AP3R     KpnI

[0132] The AP3 PCR fragment is cloned as an EcoRI, KpnI fragment betweenthe EcoRI and KpnI sites of pNosterm-bin forming pAP3-bin (FIG. 5).

EXAMPLE 3 Construction of Chimaeric Gene Fusions Between the Female(Example 2a) and Male (Example 2b) Germ-Line Specific Cassettes and theMet1 Antisense Gene.

[0133] Expression of the MET1 gene can be reduced in the female or malegerm lines by employing techniques known in the art. For example MET1down-regulation can be achieved by expressing antisense MET1 orantisense MET1 fragments or sense MET1 or partial sense MET1 orribozymes directed against MET1 or combinations of the preceding, frompromoters expressed in the required germ-line. Below is an example of anantisense MET1 approach.

[0134] a) The Construction of a Female Germ-Line Specific Met1as Gene

[0135] The MET1 cDNA is 4.7 kb long and is isolated by RT-PCR fromArabidopsis cDNA using the primers MET1F and MET1R.5′ ACTCGAGATTTTGAAAATGGTGGAAAATGGGGC 3′ MET1F      XhoI5′ ACCCGGGTGGTTATCTAGGGTTGGTGTTGAGGAG 3′ MET1R      SmaI

[0136] The resulting MET1 PCR fragment is then cloned as a SmaI, XhoIfragment between the SmaI and SalI sites of pAGL5-bin formingpAGL5-asMET1 (FIG. 6).

[0137] b) The Construction of a Male Germ-Line Specific Met1as Gene

[0138] The MET1 PCR fragment is cloned as a SmaI, XhoI fragment betweenthe SmaI and SalI sites of pAP3-bin forming pAP3-asMET1 (FIG. 7).

EXAMPLE 4 Introduction of Female and Male Germ-Line SpecificDemethylating Genes into Transgenic Plants

[0139] Chimaeric genes were introduced via Agrobacterium-mediatedtransformation into wild type diploid Arabidopsis using well knowntechniques.

[0140] a) pAGL5Met1as

[0141] Transgenic Arabidopsis plants containing the pAGL5Met1as genewere vegetatively normal and produced flowers with the normal complementof floral organs. Arabidopsis containing pAGL5Met1as were pollinatedwith pollen from wild-type diploid plants or allowed to self pollinate.Endosperm development in the resulting seeds was monitored by confocalmicroscopy (Scott et al., 1998) and seed weights were measured atmaturity. In both cases, endosperms showed a paternal excess phenotype(average maximum endosperm size=800 nuclei, delayed cellularisation(+1-2 days relative to 2x-2x crosses wild type) and chalazal endospermhyperplasia) similar to that obtained in 2x-4x crosses between wild typeplants (Table 1).

[0142] The mean weight of mature seed collected from pAGL5Met1 as plantswas 40 μg, compared with a mean of 22% g for 2x-2x seed. This representsan increase in seed weight compared to the mean of the 2x-2x.

[0143] The germination frequency was comparable to that of seed from2x-2x wild type crosses—95-100%.

[0144] The outcomes of the crosses were variable and depended on theparticular transgenic plant.

[0145] The pAGL5Met1as gene could be transformed into other crop speciessuch as B. napus and Zea mays, leading to an increase in seed size andseed quality in the transgenic plants. In this case it is mostpreferable to use MET1 and AGL5 orthologous sequences from B.napus andZea mays.

[0146] b) pAP3Met1as

[0147] A proportion of transgenic Arabidopsis plants containing thepAP3Met1as gene were vegetatively normal and produced flowers with thenormal complement of floral organs.

[0148] Arabidopsis containing pAP3Met1as were pollinated with pollenfrom wild-type diploid plants or allowed to self pollinate. Endospermdevelopment in the resulting seeds was monitored by confocal microscopy(Scott et al., 1998) and seed weights were measured at maturity. In bothcases, endosperms showed a moderate maternal excess phenotype increasedperipheral endosperm cell number, precocious cellularisation andchalazal endosperm hypoplasia qualitatively similar to that obtained in4x-2x crosses between wild type plants (Table 1).

[0149] The mean weight of mature seed collected from pAP3Met1as plantsis less than that of 2x-2x seed.

[0150] The germination frequency was comparable to that of seed from2x-2x wild type crosses—about 95-100%.

[0151] The pAP3Met1as gene could be transformed into other crop speciessuch as B. napus and Zea mays, leading to an decrease in seed size inthe transgenic plants. In this case it is most preferable to use MET1and AP3 orthologous sequences from B.napus and Z. mays.

EXAMPLE 5 Promoting Interspecific Hybridisation

[0152] Tetraploid Arabidopsis thaliana were obtained by the method,known to those skilled in the —Art, of Colchicine doubling of a diploidplant. Cross pollination between tetraploid Arabidopsis thaliana (4xA.thaliana) and Arabidopsis lyrata, results in 100% shrivelled seed(FIG. 8A) that fail to germinate (Table 3). Abortion is due to endospermfailure resulting from lethal relative genomic imbalance (FIG. 8B). Thispost-fertilisation hybridisation barrier is overcome by introducing theMet1a/s gene into the 4x A.thaliana parent; the resulting plants producehypomethylated gametes. Cross pollination between a 4x A.thaliana Met1a/s seed parent and Arabidopsis lyrata, results in plump seed (FIG. 8B)which germinate at high frequency (Table 3). This illustrates theutility of hypomethylation, as conditioned by the Met1 a/s gene in thisexample, to promote inter-specific hybridisation between two plants thatdo not normally form viable hybrids. pAGL5Met1as and pAP3Met1as weretransformed into Brassica campestris and Brassica oleraceae via standardmethods. Reciprocal crosses between the transgenic individuals of thetwo species yield plump seeds which germinate to give hybrid plants.Crosses between wild type individuals of the two species result inshrivelled seeds which fail to germinate. Hence the two transgenesovercome the normal barrier to interspecific hybridisation. The samegenes could be used in other species or varieties to promotehybridisation. TABLE 3 Relaxing genomic imprinting throughhypomethylation can promote or prevent hybrid formation Outcome of CrossEndosperm Seed viability Hybrids Cross phenotype (% germination) formed?4× A. thaliana × ME 0 NO A. lyrata 4× A. thaliana Moderate PE 95-100 YESMetIa/s × A. lyrata 4× A. thaliana × Moderate PE 95-100 YES C. arenosa4× A. thaliana × Lethal PE 0 NO C. arenosa 4× A. thaliana MetIa/s ×Lethal PE 0 NO C. arenosa

EXAMPLE 6 Preventing Interspecific Hybridisation

[0153] Cross pollination between tetraploid Arabidopsis thaliana (4x A.thaliana) and Cardaminopsis arenosa, results in 100% plump seed (FIG.9A) that germinates at high frequency (Table 3). The hybrid is asynthetic version of a naturally occurring hybrid between these twospecies Arabidopsis suesica (Chen et al., 1998). Cross pollinationbetween diploid Arabidopsis thaliana (2x A. thaliana) and C. arenosa,results in 100% shrivelled seed that fails to germinate (Table 3).Accordingly, C. arenosa can be said to have a genomic strength that issufficiently high to cause seed abortion when combined with 2x A.thaliana, but not when combined with 4x A. thaliana. To demonstrate thathypomethylation can prevent cross hybridisation between A. thaliana andC. arenosa the Met1 a/s gene was introduced into 4x A. thaliana, andthis plant used as seed parent in a cross to C. arenosa. Seed from sucha cross is 100% shrivelled (FIG. 9B) and fails to germinate (Table 3).The same gene could be used in other species or varieties to prevent theproduction of viable hybrid seed.

EXAMPLE 7 Maternal Hypomethylation Promotes Autonomous EndospermDevelopment

[0154] In the absence of fertilisation, Arabidopsis plants heterozygousfor the fie-1 mutation (fie/FIE) produce seeds with partial endospermdevelopment (Ohad et al., 1996; 1999; see also Table 3 and FIGS. 12A-C).These ‘autonomous’ endosperms consist of a severely reduced number ofendosperm nuclei (compared to wild type controls) and the endospermfails to undergo cellularisation. The seed collapses and becomesshrivelled at maturity (Table 4). Consequently, the fie mutationconditions only limited endosperm development restricting its utility inthe production of autonomous apomictic seed crops or embryoless seedcrops. Endosperms produced in plants carrying the fis1/mea and fis 2mutations are very similar to those of fie/FIE plants, and hence theutility of these genes is also restricted.

[0155] Since “fie” endosperms do not contain a paternal genomiccontribution one hypothesis is that proper development of the endospermrequires the expression of paternally derived genes that are subject tomaternal imprinting.

[0156] When plants heterozygous for the fie mutation are pollinated withwild type pollen from a 2x wild type plant the ovules carrying the fieallele develop into seeds that abort at heart/torpedo stage, whileovules carrying the wild type FIE allele develop normally (Ohad et al.,1996; 1999; Table 4 and FIG. 10). The aborted seeds express a strongpaternal excess phenotype (Table 4; FIG. 10), despite containing only asingle paternal contribution. This suggests that a complex situationwith respect to imprinting applies within fertilised and unfertilisedfie endosperms. One hypothesis is that the fie mutation lifts imprintingfrom a proportion of genes normally subject to maternal imprinting: theintroduction of a additional paternal genome following fertilisationgenerates an effective lethal paternal excess such as encountered in a2x-6x wild type cross (Table 1). The failure of fie endosperms todevelopment normally in the absence of fertilisation is also accountedfor by this hypothesis, since not all maternally imprinted genes may bederepressed.

[0157] Since gametes derived from hypomethylated plants (Met1as and ddm)appear to have no or highly attenuated imprinting, and therefore act inpart as gametes of the opposite sex in endosperms, we hypothesised thatsuch gametes in combination with the fie mutation would promote completeendosperm development. In the first experiment, we used pollen from aMet1as plant [FIE/FIE; MET1 als/MET1 a/s] to pollinate a FIE/fieheterozygote [fie/FIE; METI als/MET1 a/s] and found most seeds producedwere plump and viable (Table 4; FIG. 11). The seeds segregate 1:1 forthe FIE/FIE:FIE/fie genotypes, showing that the fie allele istransmissible through the seed parent in this cross. The FIEFIE seedsdisplay a maternal excess phenotype as expected—endospermunder-development (Table 5) and a reduced seed weight (Table 4), whilstthe Fiefie seeds display a moderate paternal excess phenotype (Table 5),similar to that observed in a 2x×4x cross between wild type A. thalianaplants. When wild type pollen from a diploid plant is used in thiscross, the resulting seeds segregate 1:1 forplump/viable:shrivelled/inviable and the ovules containing the fiemutation produce inviable seed since the plump seeds all contain thewild type FIE allele (Table 4; FIG. 10). The abortive seeds display apaternal excess phenotype similar to that observed in a 2x-6x crossbetween wild type A. thaliana plants (FIGS. 3 and 10; Table 5).Therefore, paternal gametes from Met1as plants appear to rescue fiecontaining seeds from lethality by reducing the magnitude of thepaternal excess phenotype. This supports the hypothesis as outlinedabove.

[0158] In the second experiment we combined the fie mutation and theMet1as gene into the same individual (see Table 4 and FIG. 12). Whenthese plants were emasculated and left unpollinated 50% of the ovulesunderwent autonomous endosperm development as expected for ovulescarrying the fie mutation. Confocal microscopy showed that these seedscontain well developed, cellularised endosperms (FIG. 12), with between500-700 peripheral nuclei, a cellularisation time of 5-8 days and avolume of chalazal endosperm between 0.01 and 10x that of a seedproduced in a 2x-2x cross. The mature seeds were shrivelled, but weighed15 μg. In contrast, developing ovules of emasculated and unpollinatedFie/fie plants contain very under-developed endosperm that do notcellularise (FIG. 12). These seeds contain about 200 peripheralendosperm nuclei and no recognizable chalazal endosperm. The matureseeds were shrivelled and weighed 5 μg. The production of an endospermthat has the main features of a wild type endosperm (numerous peripheralendosperm nuclei, cellularisation, and a chalazal endosperm) in plantscontaining both the fie mutation and the Metal as gene shows that thelifting or attenuation of imprinting within the maternal gamete asconditioned by the Met1as gene is sufficient to relieve thedevelopmental block encountered in unpollinated fie ovules. This greatlyextends the utility of the autonomous endosperm mutants (fis1, fis2,fis3, and fie). TABLE 4 Enhancement of endosperm development in fiemutant seeds by hypomethylation Mature Extent of seed phenotypesendospermn (%)¹ Seed viability² Seed weight μg)³ development PlumpShrivelled Plump Shrivelled Plump Shrivelled (%)⁴ seeds seeds seedsseeds seeds Seeds Complete Partial FIE/fie X 50 50 95-100 0 25 15 50 50⁵ 2x FIE/fie X 100 0 95-100 NA 50% = 15 NA 100  0  2xmet 50% = 30FIE/fie 0 100 NA 0 NA  5 0 100⁶ emasculate FIE/fie: 0 100 NA 0 NA 20 100 0  2xmetHET emasculate

[0159] TABLE 5 Endosperm development in crosses involving fie, met1a/sand wild type plants. fie/FIE × FIE/FIE met/met fie/FIE × FIE/FIEFIE/FIE fie/FIE FIE/FIE fie/FIE seeds seeds seeds seeds Maximum numberof 192 637 447 408 P.E nuclei Timing of endosperm 3-4 DAP 7-8 DAP 5-6DAP >10 DAP cellularisation Size of chalazal 0.05-0.1× 3-4× 1× 10-15×Endosperm¹

EXAMPLE 8 Production of Plants that Combine the fie Mutation and theFemale Germ-Line Specific Demethylating Gene, AGL5Met1a

[0160] Plants heterozygous for the fie mutation and hemizygous for thepAGL5Met1as gene were generated by making crosses between FIE/fie plantsas pollen parent and plants homozygous for the pAGL5Met1as gene as seedparent. These plants were vegetatively normal and produced normalflowers. When emasculated 50% of the ovules initiated seed developmentwithout fertilisation. Confocal microscopy showed that endospermdevelopment was extensive, resulting in a large (500-700 nuclei)cellularised endosperm.

[0161] The pAGLSMet1as gene could be introduced into crop species, suchas B.napus and Zea mays in which expression of the FIE gene, or any ofthe genes that condition autonomous endosperm development, is suppressedor absent through mutation or the use of transgenic technologies, toproduce promote apomixis or embryoless (pseudoapomictic) seed.Preferably the pAGL5Met1as construct contains B.napus or Z.mays MET1 andAGL5 orthologous sequences

EXAMPLE 9 The Use of FIE Down-Regulation to Paternalise Female Gametes(Polar Nuclei)—to Increase Endosperm Size and Seed Weight.

[0162] When plants heterozygous for the fie mutation (Ohad et al., 1996;1999) are pollinated with pollen from a 2x wild type plant the ovulescarrying the fie allele develop into seeds that abort at heart/torpedostage, while ovules carrying the wild type FIE allele develop normally(Ohad et al, 1996; 1999; Table 4 and FIG. 10). The aborted seeds expressa strong paternal excess phenotype (Table 4; FIG. 10), despitecontaining only a single paternal contribution. This suggests that acomplex situation with respect to imprinting applies within fertilisedand unfertilised fie endosperms. This is explained by proposing that thefie mutation lifts imprinting from genes normally subject to maternalimprinting (the maternal gametes are thus strongly paternalised): theintroduction of a additional paternal genome following fertilisationgenerates an effective lethal paternal excess (2maternal;3paternal) suchas encountered in a 2x-6x wild type cross (2m::3p) (Table 1).

[0163] Since gametes derived from hypomethylated plants (Met1as and ddm)appear to have no or highly attenuated imprinting, and therefore act inpart as gametes of the opposite sex in endosperms, such gametes incombination with the fie mutation could promote complete endospermdevelopment. In the first experiment, pollen from a Met1as plant[FIE/FIE; METI a/s/METI a/s] is used to pollinate a FIE/fie heterozygote[fie/FIE; METI a/s/METI a/s] and most seeds produced were plump andviable (Table 4; FIG. 11). The seeds segregate 1:1 for theFIE/FIE:FIE/fie genotypes, showing that the fie allele is transmissiblethrough the seed parent in this cross. The FIEFIE seeds display amaternal excess phenotype as expected—endosperm under-development (Table5) and a reduced seed weight (Table 4), whilst the Fiefie seeds displaya moderate paternal excess phenotype (Table 5), similar to that observedin a 2x×4x cross between wild type A. thaliana plants. When wild typepollen from a diploid plant is used in this cross, the resulting seedsegregate 1:1 for plump/viable:shrivelled/inviable and the ovulescontaining the fie mutation produce inviable seed since the plump seedsall contain the wild type FIE allele (Table 4; FIG. 10). The abortiveseeds display a paternal excess phenotype similar to that observed in a2x-6x cross between wild type A. thaliana plants (FIGS. 3 and 10; Table5). Therefore, paternal gametes from Met1as plants appear to rescue fiecontaining seeds from lethality by reducing the magnitude of thepaternal excess phenotype. As the fie mutation appears to cause strongpaternalisation of the maternal gametes (polar nuclei), wild-type FIEmay participate directly in maternal imprinting (as part of theimprinting complex).

[0164] The paternalisation of the polar nuclei by the fie mutation ismore extensive than that achieved by metIa/s since a fie×2x crossresults in lethal paternal excess (Table 4; FIG. 10), but a met1a/s×2xcross produces viable paternal excess, with increased endosperm size andseed weight (Table 1). Thus the degree of paternalisation of the polarnuclei determines the outcome of crosses with pollen from diploid wildtype plants: moderate paternalisation (e.g Met1a/s) produces a largeviable seed due to moderate paternal excess in the endosperm, whereasstrong paternalisation (e.g fie null mutation) results in seed lethalitydue to excessive paternal excess in the endosperm. Modulating FIEexpression may have application in manipulating endosperm size and seedweight. The fie mutation used is a null allele (fie-1; Ohad et al,1999)— no functional FIE protein is produced, resulting in strongpaternalisation of the polar nuclei, and seed lethality in crosses withwild type pollen from a diploid plant. Reducing, but not eliminating theexpression of FIE results in moderate paternalisation of the polarnuclei; the exact level of paternalisation being directly related to theamount of FIE protein expression during female gametogenesis. Reductionin FIE expression can be achieved using a number of well known methodssuch as antisense RNA expression against the sense FIE RNA transcript.Incremental reduction in FIE expression, by making use of for exampledifferent, more or less effective, anti-sense lines, identifies a levelof FIE expression that is optimal for producing viable seeds with amaximally increased endosperm size and seed weight.

[0165] Suitable anti-sense genes would comprise the FIE promoter drivingtranscription of the anti-sense FIE transcribed region. Other genessuitable to reduce the levels of FIE expression and deliver levels ofpaternalisation of polar nuclei intermediate between a FIE null alleleand the wild type FIE allele include genes encoding fragments of the FIEprotein which recognise and bind to-imprinted genes, but are ineffectivein promoting their non-expression in the endosperm (e.g. because therepressive complex cannot form or cannot be maintained).

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1 6 1 34 DNA Artificial Sequence Description of Artificial SequencePrimer 1 ccgaattctt caagcaaaag aatctttgtg ggag 34 2 34 DNA ArtificialSequence Description of Artificial Sequence Primer 2 cggtacctataagccctagc tgaagtataa acac 34 3 37 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 3 ccgaattcaa gcttcttaag aattatagta gcacttg37 4 39 DNA Artificial Sequence Description of Artificial SequencePrimer 4 gggtaccttc tctctttgtt taatcttttt gttgaagag 39 5 33 DNAArtificial Sequence Description of Artificial Sequence Primer 5actcgagatt ttgaaaatgg tggaaaatgg ggc 33 6 34 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 acccgggtgg ttatctagggttggtgttga ggag 34

1. A method for the production of modified endosperm, which comprisesthe step of transforming a plant, or plant propagating material, with anucleic acid molecule comprising one or more regulatory sequencescapable of directing expression in the male or female germ line and/orgametes of the resultant plant, and one or more sequences whoseexpression or transcription product(s) is/are capable of modulatinggenomic imprinting.
 2. A method for the production of modifiedendosperm, which comprises the step of transforming a plant, or plantpropagating material, with a nucleic acid molecule comprising one ormore regulatory sequences capable of directing expression within thedeveloping gynoecium, especially the cell lineage that gives rise to orcomprises the female germ line (megasporocyte tissue), within the ovuleof the resultant plant, and one or more sequences whose expression ortranscription product(s) is/are capable of modulating genomicimprinting.
 3. A method for the production of modified endosperm whichcomprises the step of transforming a plant, or plant propagatingmaterial, with a nucleic acid molecule comprising one or more regulatorysequences capable of directing expression within the developing stamen,especially the cell lineage that gives rise to or comprises the malegerm line (microsporocyte tissue) of the resultant plant and one or moresequences whose expression or transcription product(s) is/are capable ofmodulating genomic imprinting.
 4. A method as claimed in any one ofclaims 1 to 3 wherein the nucleic acid molecule comprises one or moresequences whose expression or transcription product(s) is/are associatedwith the formation and/or maintenance of genomic imprints.
 5. A methodas claimed in claim 4 wherein the nucleic acid molecule includes asequence encoding a histone deacetylase, methyl cytosine binding proteinor Sin 3 protein.
 6. A method as claimed in any one of claims 1 to 5wherein the nucleic acid molecule includes a sequence of the FIE gene orFIS genes.
 7. A method as claimed in any one of claims 1 to 6 whereinthe nucleic acid molecule comprises one or more sequences whoseexpression or transcription product(s) is/are capable of altering thedegree of methylation of nucleic acid.
 8. 8. A method for the productionof modified endosperm, which comprises the step of transforming a plant,or plant propagating material, with a nucleic acid molecule comprisingone or more regulatory sequences capable of directing expression in themale or female germ line and/or gametes of the resultant plant, and oneor more sequences whose expression or transcription product(s) is/arecapable of altering the degree of methylation of nucleic acid.
 9. Amethod for the production of modified endosperm, which comprises thestep of transforming a plant, or plant propagating material, with anucleic acid molecule comprising one or more regulatory sequencescapable of directing expression within the developing gynoecium,especially the cell lineage that gives rise to or comprises the femalegerm line (megasporocyte tissue), within the ovule of the resultantplant, and one or more sequences whose expression or transcriptionproduct(s) is/are capable of altering the degree of methylation ofnucleic acid.
 10. A method for the production of modified endospermwhich comprises the step of transforming a plant, or plant propagatingmaterial, with a nucleic acid molecule comprising one or more regulatorysequences capable of directing expression within the developing stamen,especially the cell lineage that gives rise to or comprises the malegerm line (microsporocyte tissue) of the resultant plant and one or moresequences whose expression or transcription product(s) is/are capable ofaltering the degree of methylation of nucleic acid.
 11. A method asclaimed in any one of claims 1 to 10 wherein the one or more regulatorysequences comprise a promoter sequence, or regulatory sequences orfragments therefrom.
 12. A method as claimed in any one of claims 1, 2,4 to 9, or 11 wherein the promoter is derived from the Arabidopsis AGL5gene, the Petunia FBP7, the Petunia FBP 11 gene, the Arabidopsis BEL1gene, the Arabidopsis MEDEA (FIS1) gene, the Arabidopsis FIS 2 gene, theArabidopsis FIE (FIS 3) gene, orthologs/homologues of any of these genesfrom other species or any promoter that drives expression that isrestricted to cells within the female reproductive organs thatcontribute to the female germ line, preferably promoters fromgynoecium-specific genes that are first expressed during early gynoeciumdevelopment, preferably before the differentiation of individual ovules,and which maintain their expression until ovule differentiation iscomplete.
 13. A method as claimed in any one of claims 1, 3, 4 to 8, 10or 11 wherein the promoter is derived from the Arabidopsis geneAPETALA3, the Arabidopsis PISTILATTA gene, the Arabidopsis E2 gene, theArabidopsis APG gene, homologues/orthologs of these genes from otherspecies or any promoter that drives expression that is restricted tocells within the male reproductive organs that contribute to the malegerm line, preferably promoters from stamen-specific genes that arefirst expressed during early stamen development, preferably before thedifferentiation of individual microsporocytes, and which maintain theirexpression until stamen differentiation is complete.
 14. A method asclaimed in any one of claims 1 to 13 wherein the size of the endospermis altered.
 15. A method as claimed in any one of claims 1 to 13 whereindevelopment of the endosperm is altered.
 16. A method as claimed in anyone of claims 7 to 15 wherein the degree of nucleic acid methylation isincreased.
 17. A method as claimed in claim 16 wherein the nucleic acidmolecule includes a sequence encoding a methylating enzyme such asMethylase 1, Methylase 1-like enzyme, Methylase 2 or Chromomethylase ofArabidopsis.
 18. A method as claimed in any one of claims 7 to 15wherein the degree of nucleic acid methylation is decreased.
 19. Amethod as claimed in claim 18 wherein the nucleic acid molecule includesa sequence encoding a de-methylating enzyme such as de-methylase(=MeCP2homologue) of Arabidopsis.
 20. A method as claimed in claim 18wherein reduction in nucleic acid methylation is achieved bydown-regulation of one or more methylating enzymes present in the plant.21. A method as claimed in claim 20 wherein the nucleic acid moleculeincludes a sequence encoding an antisense nucleic acid molecule, a fullor partial copy of a methylating enzyme gene already present in theplant or sequence, or a sequence encoding a ribozyme.
 22. A method asclaimed in claim 21 wherein the nucleic acid molecule includes thesequence of the Met1as gene.
 23. An isolated or recombinant nucleic acidmolecule, eg a DNA molecule, which comprises one or more regulatorysequences capable of directing expression in the male or female germline and/or gametes of a plant and one or more sequences whoseexpression or transcription product(s) is/are capable of altering thedegree of methylation of nucleic acid.
 24. A nucleic acid molecule asclaimed in claim 23 wherein the degree of nucleic acid methylation isdecreased.
 25. The use of a nucleic acid molecule as claimed in claim 24as a barrier to hybridisation between plants.
 26. The use as claimed inclaim 25 wherein the plants are of the same species.
 27. The use asclaimed in claim 25 or claim 26 wherein the barrier results from failurein endosperm development.
 28. The use of a nucleic acid molecule asclaimed in claim 24 in overcoming a barrier to hybridisation betweenplants.
 29. The use as claimed in claim 28 wherein the plants are of thesame species.
 30. The use as claimed in claim 28 or claim 29 wherein thebarrier results from failure in endosperm development.
 31. A nucleicacid molecule as claimed in claim 23 modified by any one or more of thefeatures defined in any one of claims 12 to
 22. 32. A nucleic acidmolecule as claimed in any one of claims 23 to 31 which is in the formof a vector.
 33. A plant cell including nucleic acid as defined in anyone of claims 23 to
 32. 34. A transgenic plant (or parts thereof such aspropagating material) including nucleic acid as defined in any one ofclaims 23 to
 33. 35. A method for modulating genomic imprinting inplants, which comprises the step of transforming a plant, or plantpropagating material, with a nucleic acid molecule comprising one ormore regulatory sequences capable of directing expression in the male orfemale germ line and/or gametes of the resultant plant, and one or moresequences whose expression or transcription product(s) is/are capable ofaltering the degree of methylation of nucleic acid.